The hydrolysis of fats, also known as fat splitting, has long been accomplished by the use of high pressure steam. Steam splitter reaction conditions are typically about 250.degree. C. and 750 psig. To maintain these conditions a boiler is required to supply the high pressure steam as well as sophisticated pumps capable of pumping feedstock and water into the steam splitting column at high pressure. The costs involved for this type of operation, as required for capital investment as well as process costs such as for energy in the forms of steam, natural gas and electricity, are of course, very high.
There is thus a significant economic incentive to develop new more efficient processes for the hydrolysis of fats, and, as has already been demonstrated in Japan, enzymatic fat splitting is clearly the process of choice. The enzyme that catalyzes the hydrolysis of fats is called lipase, or more formally E.C. 3.1.1.3 glycerol-ester hydrolase. The overall chemistry of the reaction is shown below for a typical triglyceride: ##STR1##
It may be noted that the above reaction actually proceeds via stepwise hydrolysis of the acyl groups on the glyceride, so that any given time, the reaction mixture contains not only triglyceride, water, glycerol, and fatty acid, but also diglycerides and monoglycerides. Furthermore the reaction is reversible. The reverse reaction between an alcohol and a fatty acid to form an ester is called esterification. To force the forward reaction to completion, it is necessary to remove one of the products from the reaction mixture. This task is made easy by the fact that the reaction actually takes place in a biphasic medium. The triglyceride and fatty acid form an oil layer, while the water and glycerol form an aqueous layer. Thus the hydrolysis is easily forced to completion in the splitter by removing the glycerol in the sweetwater.
Lipase can be isolated from several sources soil, plants, animals, or microorganisms. However, there are important differences in the substrate specificity of the lipases harvested from different sources. For example, porcine pancreatic lipase is position specific for the terminal (sn-1 and sn-3) ester bonds on the triglyceride. Lipase from several microbes, such as Rhizopus arrhizus and Mucor miehei also show the same positional preference for the end acyl groups. Another type of specificity is exhibited by the lipase secreted by Geotrichum candida. This lipase preferentially liberates unsaturated fatty acid groups containing a cis double bond in the 9-position of the acyl group, such as oleic and linoleic acids.
A third type of substrate specificity shown by some lipases is that of chain length. Pregastric esterases of lamb, goat, and kid selectively split shorter carbon chain length acyl groups (C.sub.4 -C.sub.8). These lipases, are used in the manufacture of Italian cheeses. The distinctive flavor of these cheeses is caused by the release of short chain fatty acids. Lipase from Aspergillus niger has also shown a similar specificity for shorter chain lengths.
Lipases that show no substrate specificity and are thus random in their attack on the glyceride molecule also exist. This is the type of enzyme catalyst that is needed for the fat splitting reaction. The most prevalent nonspecific lipase is isolated from the yeast Candida cylindracea, which has been reclassified recently to Candida rugosa. Pseudomonas type bacteria have also been found to excrete a nonspecific lipase.
There are many references concerned with enzymatic fat hydrolysis. In Household & Personal Products Industry, August 1981, Page 31, there was disclosed the use of lipase in an enzymatic process in which oil and fat are separated into fatty acid and glycerine. Likewise, the following references discuss various methods of effecting enzymatic fat hydrolysis:
A. Mitsutani, Research & Development Review Report No. 27, Application of Microbiological Technology to Chemical Process, Nippon Chemtec Consulting, Inc., March 1984. PA0 W. Linfield, R. Barauskas, L. Sivieri, S. Serota, R. Stevenson, "Enzymatic Fat Hydrolysis and Synthesis", JAOCS, 61 (2), February 1984, pp. 191-195. PA0 W. Linfield, D. O'Brien, S. Serota, R. Barauskas, "Lipid-lipase Interactions I. Fat Splitting with Lipase from Candida rugosa", JAOCS, 61 (6), June 1984, pp. 1067-1071. PA0 G. Benzonana, S. Esposito, "On the Positional and Chain Specificities of Candida Cylindracea Lipase", Biochim. Biophys. Acta, 231 (1971) pp. 15-22. PA0 a. a first layer consisting of a hydrophobic filter cloth having openings from about 3 to about 5 microns in size; PA0 b. a second layer adjacent to said first layer comprising fibers of a hydrophobic microporous thermoplastic polymer selected from the group consisting of aliphatic olefinic polymers, oxidation polymers, ionic polymers and blends thereof, having lipase immobilized on the fibers by adsorption from an aqueous solution either without pretreatment or following pretreatment of the fibers only by wetting with a polar water miscible organic solvent in which the polymer is insoluble and which does not deactivate the lipase; and PA0 c. a third layer adjacent to the side of said second layer opposite said first layer comprising a retaining means capable of maintaining the fibers of the second layer in place.
None of the above references discusses the use of immobilized enzymes. The immobilization of enzymes on solid supports has advantages that have long been recognized. A particular advantage is that the immobilized enzyme remains bonded to the support rather than passing through with the substrate upon which it is acting so that there is no need to recover the enzyme from the substrate and so that the enzyme remains in the support where it may be reused.
Japanese Patent Publication No. JP 84091883 (Abstract No. 84-168208) of May 5, 1984 discloses that an immobilized enzyme may be produced by bringing an aqueous solution of enzyme into contact with a porous synthetic hydrophobic adsorbent. Examples given to adsorbent materials are styrene and methacrylic acid ester. The reference, however, gives no hint to the hydrolysis of fats, nor to lipase as the enzyme.
Russian Patent Publication No. SU 804647 (Abstract No. 83249D) of Feb. 15, 1981 discloses crosslinked porous styrene polymers used as activity enhancing carriers for immobilized enzymes, but also does not hint to the composition, process, methods or apparatus of the present invention.
There is also art that teaches the hydrolysis of fats by use of immobilized lipase. In Chemical Week, Vol. 133, No. 22, Nov. 30, 1983, on page 33, it is generally mentioned that a number of useful enzymes may be immobilized by locking them to a carrier by adsorption, crosslinking or covalent bonding, and on page 34 there is mention that lipase may be used to hydrolyze fat, but there is no teaching in this reference of polymeric carriers, and there is a warning on page 33 that an enzyme free in solution and the same enzyme locked to a carrier do not behave the same.
In J. Lavayre, J. Baratti, "Preparation and Properties of Immobilized Lipases", Biotech & Bioengr., 24 (1982), pp. 1007-1013, hereinafter referred to as "Lavayre et al", there is discussed the use of lipase immobilized by adsorption onto a hydrophobic support for the hydrolysis of olive oil. The Lavayre et al article, however, states that when purified pancreatic lipase was used, the specific activity of the immobilized enzyme was 17 to 25% that of the soluble enzyme. Furthermore, the only support used in the hydrolysis tests was the iodopropyl derivative of porous glass (Spherosil).
The use of lipase immobilized onto polyacrylamide beads for the hydrolysis of triglyceride is discussed in "Bell, Todd, Blain, Paterson and Shaw", Hydrolysis of Triglyceride by Solid Phase Lipolytic Enzymes of Rhizopus arrhizus in Continuous Reactor Systems", Biotech & Bioengr., 23 (1981), pp. 1703-1719, and in Lieberman and Ollis, "Hydrolysis of Particulate Tributyrin in a Fluidized Lipase Reactor", Biotech & Bioengr., 17 (1975), pp. 1401-1419. In those references, however, the immobilization is effected by covalent bonding (e.g. diazonium intermediate), not adsorption. The results were a significant decrease in the activity of the immobilized as compared to the free enzyme.
The hydrolysis of fats with lipase is a reversible reaction and there are teachings in the art of methods of producing fats by reacting a fatty acid with water and glycerol in the presence of lipase. One such reference is M. M. Hoq, T. Yamane, S. Shimizu, T. Funada, S. Ishida, "Continuous Synthesis of Glycerides by Lipase in Microporous Membrane Bioreactor", JAOCS, 61 (4), April 1984, pp 776-781, hereinafter referred to as "Hoq et al". Hoq et al advises against the use of immobilized lipase for the stated reason that its activity is commonly only several percent of the original activity of the free lipase. Hoq employs a device, it refers to as a bioreactor, which comprises supported hydrophobic microporous membrane, in particular one made from polypropylene, that is placed at the interface of an upper phase of fatty acid and lower phase of a solution of glycerol, water and lipase. The reactants and lipase come into contact at the interface of the two phases thereby causing the reaction, the glycerides diffusing back into the bulk flow of the fatty acid phase.
The present invention is based on the surprising discovery that lipase immobilized on certain porous polymeric supports in a certain manner loses very little of its fat hydrolysis activity as compared to soluble lipase, notwithstanding teachings of prior art such as Lavayre et al and Hoq et al that immobilization of lipase causes such activity to diminish to a small fraction of the free lipase.